Although it is well-known to express both prokaryotic and eukaryotic non-native (“heterologous”) proteins in microbial hosts, there are a number of technical difficulties which must be overcome in expressing a protein; these include (i) attaining a sufficient level of expression, (ii) obtaining the protein in a form which is readily isolated and purified, and (iii) ensuring that the protein is fully expressed and not truncated as by incomplete translation or by subsequent degradation. One approach has been to co-express the protein of interest with another “carrier” protein by joining the genes for each; the resulting fusion proteins usually contain the two gene products covalently linked together, either directly or through a short linking segment.
A particularly favorable expression system has been described in U.S. Pat. No. 5,124,255, which patent is expressly incorporated herein by reference. In that patent, fusion proteins are disclosed which utilize as a carrier protein the Escherichia coli enzyme CKS (CMP-KDO synthetase, or CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase). That carrier permits the expression at high levels of the desired fusion proteins, in which the protein of interest is encoded for by DNA which is positioned at the 3′ end of the carrier gene. However, it can occur in this and other expression systems that the fusion product is not easily separated from closely-related expression products or cellular contaminants of similar molecular weight. Also, it may happen that the protein of interest, which typically is at the C-terminus of the fusion protein, is truncated rather than being full-length.
These difficulties must be overcome when, for example, the protein of interest is an epitope or combination of epitopes which is to be used in a diagnostic immunoassay, and consequently must be fully-expressed and purifiable. It therefore remains an object of the present invention to develop improved expression systems which permit the reliable expression of a wide variety of complete heterologous proteins, and furthermore which facilitate the purification of such proteins.